Targeting eEF2 with the protein translation elongation inhibitor SVC112 in head and neck squamous cancer

摘要放射治疗（RT）和PD-1抑制剂的免疫治疗用于早期和复发性帕金森病。 头颈部鳞状细胞癌（HNSCC）; RT失败很常见，大多数患者都是如此 对PD-1抑制没有反应。两者都影响肿瘤微环境（TME），但对TME的研究有限 人类免疫细胞模型的可用性。PI 3 K/mTOR信号通过调节蛋白质合成 转录因子如Myc或SOX 2，其决定HNSCC中的生长、侵袭和耐药性。SVC112 是一种完全合成的小分子，通过抑制eEF 2在延伸步骤抑制蛋白质合成。SVC112 对癌细胞的作用强于非癌细胞，比高三尖杉酯碱更有效，选择性更强 (HHT)FDA批准的翻译延长抑制剂。癌细胞比非癌细胞具有更高的eEF 2， 最敏感品系eEF 2表达量最高。SVC 112耗尽SOX 2、Myc和细胞周期蛋白D1 在HNSCC细胞中，浓度对蛋白质组的其余部分影响最小。SVC 112降低 在体外的球，减少体内肿瘤生长的患者来源的异种移植物（PDX），并诱导肿瘤消退 当在四个PDX模型中的三个中给予RT时。SVC 112还降低了人类中E6癌蛋白的表达。 乳头瘤病毒驱动的细胞。最后，SVC 112降低PD-1配体（PD-L1），导致PD-L1：PD-1降低 这种相互作用导致球形T细胞侵袭增加，同时对T细胞适应性或功能没有影响。因此，在本发明中， SVC 112可以靶向多个关键通路（SOX 2、Myc、PD-L1），并且可以影响TME以逆转免疫应答。 逃避为了测试SVC 112在HNSCC中的治疗潜力，将使用几种独特的工具，包括1）球体 含有癌细胞和T细胞（iSpheres），能够在体外研究TME相互作用和免疫治疗， 2)HNSCC的同基因小鼠模型，和3）由PI开发的人源化小鼠（HM）模型， 研究TME相互作用和体内免疫疗法。SVC 112的区分作用是 由于关键蛋白质的选择性耗竭，将通过核糖体分析（以鉴定mRNA靶）进行测试， 蛋白质组学分析（以识别蛋白质靶点）。然后，我们将测试SVC 112与RT协同作用的机制， 以及SVC 112对TME的调节是否将进一步增强RT功效。最后，假设 HNSCC中SVC 112减少PD-L1将改变肿瘤-免疫相互作用，将通过定义 SVC 112对TME、T细胞肿瘤浸润和癌症免疫的关键蛋白质表达的影响 串话将检查SVC 112对T细胞适应性和功能的影响，帮助SVC 112翻译。我们 将评估iSphere和HM模型中的SVC 112和PD-1抑制，并在两者均 加起来最后，我们将测试eEF 2在SVC 112易感性中的预测价值， 在人肿瘤中的表达，以能够使用HNSCC组织鉴定SVC 112的伴随生物标志物 有1,250个病人的银行。该项目将推动在科罗拉多发现的药物SVC 112的翻译， 通过剖析其作用的基础，研究其毒性，并测试预测生物标志物以帮助临床试验。

SUMMARY. Radiation therapy (RT) and immunotherapy with PD-1 inhibitors are used in early and relapsed head and neck squamous cell cancers (HNSCC), respectively; RT failures are common, and most patients do not respond to PD-1 inhibition. Both impact the tumor microenvironment (TME), but studying the TME is limited by the availability of models with human immune cells. PI3K/mTOR signaling regulates protein synthesis through transcription factors such as Myc or SOX2, that dictate growth, invasion, and drug resistance in HNSCC. SVC112 is a fully synthetic small molecule that inhibits protein synthesis at the elongation step by inhibiting eEF2. SVC112 had greater effect on cancer over non-cancer cells, and was more potent and selective than homoharringtonin (HHT), an FDA-approved translation elongation inhibitor. Cancer cells had higher eEF2 than non-cancer cells, and the most susceptible strain had the highest eEF2 expression. SVC112 depleted SOX2, Myc, and Cyclin D1 in HNSCC cells at concentrations that had minimal effect on the rest of the proteome. SVC112 decreased spheres in vitro, reduced tumor growth in vivo in patient-derived xenografts (PDX), and induced tumor regression when given with RT in three out of four PDX models. SVC112 also reduced the E6 oncoprotein in human papillomavirus-driven cells. Lastly, SVC112 decreased PD-1 ligand (PD-L1) resulting in decreased PD-L1:PD-1 interactions leading to increased sphere T cell invasion, while having no effect on T cell fitness or function. Thus, SVC112 can target multiple key pathways (SOX2, Myc, PD-L1), and can influence the TME to reverse immune evasion. To test SVC112’s therapeutic potential in HNSCC, several unique tools will be used including 1) spheres containing cancer cells and T cells (iSpheres) that enable studying TME interactions and immunotherapy in vitro, 2) syngeneic mouse models of HNSCC, and 3) humanized mouse (HM) models, developed by the PI, that enable studying TME interactions and immunotherapy in vivo. The hypothesis that SVC112’s discriminating effect is due to selective depletion of key proteins will be tested by ribosome profiling (to identify mRNA targets) and proteomics analysis (to identify proteins targets). Then, we will test the mechanism of SVC112 synergy with RT, and if the modulation of the TME by SVC112 will further enhance RT efficacy. Lastly, the hypothesis that reduction of PD-L1 by SVC112 in HNSCC will alter the tumor-immune interaction will be tested by defining the effect of SVC112 on the expression of proteins critical for the TME, T cell tumor infiltration, and cancer-immune crosstalk. The effect of SVC112 on T cell fitness and function will be examined, aiding SVC112 translation. We will assess SVC112 and PD-1 inhibition in iSphere and HM models, and explore their efficacy when both are combined. Lastly, we will test the predictive value of eEF2 in SVC112 susceptibility, and we will assess eEF2 expression in human tumors, to enable identifying a companion biomarker to SVC112, using a HNSCC tissue bank with 1,250 patient cases. This project will propel the translation of SVC112, a drug discovered in Colorado, by dissecting the basis for its effect, studying its toxicity, and testing predictive biomarkers to aid clinical trials.